Method of inhibiting platelet aggregation

ABSTRACT

A method of inhibiting platelet aggregation using 2-bis( Beta hydroxyethyl)amino-4,5-diphenyloxazole as the anti-aggregating agent.

United States Patent Caprino July 15, 1975 METHOD OF INHIBITING PLATELET OTHER PUBLICATIONS AGGREGATION Caprmo et al., Reprint from Arzne1m.Forsch. (Drug [75] Inventor: Luciano Caprlno, Rome, Italy Res 23, 9, 12834287 (1973) [73] Assignee: lstituto Farmacologico Serono Caprino et aL, Reprint from Arzneim.-Forsch. (Drug S.p.A., Rome, Italy Res.) 23, Nr. 9, 1277-1283 (1973).

22 F d: M 1 1974 1 16 at Primary ExaminerStanley J. Friedman [21] App]. No.: 447,275 Attorney, Agent, or FirmOstr0lenk, Faber, Gerb &

Soffen [52] US. Cl. 424/272 [51] Int. Cl. A61K 31/42 [57] ABSTRACT Fleld 0f Search A method of platelet gg g using bis(,B-hydr0xyethyl)amin0-4,S-diphenyloxazole as the [56] References Cited ami aggregating agent.

UNITED STATES PATENTS 1/1971 Marchetti 260/307 R 5 Ciaims, No Drawings 1 METHOD OF INHIBITING PLATELET AGGREGATION DETAILED DESCRIPTION OF THE INVENTION adrenaline induced platelet aggregation without showing the? undesirable side effects of most of the antiinflammatory drugs.

I have now found that the compound 2-b is(B- 1 This invention relates to a method of inhibiting plate- 5 hydroxyerhyl) amino 4,5 diphenyloxazele, having the E1 aggreganonstructure:

The interest in drugs capable of inhibiting platelet aggregation is mainly related to the discovery that plate- N lets play an important role in thromboembolic disease. I The arterial thrombus mainly consists of platelets and 10 l fibrin strands For this reason, the anticoagulants, al- \O/ CH CH OH though effective in the treatment of venous thrombotic 2 2 S El h z k i g A previlntmg Previously disclosed as an anti-inflammatory drug, is ar ena mm n e m er t 8 pre effective in inhibiting platelet aggregation when tested nance of platelet? type of Clot h 15 in vitro on the ADP, collagen and adrenaline induced gbested that g g f l fumftlon 2 platelet aggregation in rabbit platelet-rich plasma. The 4 c more use u t an t 6 Stan at mucoagu ant t same activity has been shown by the above compound g th h b t n h th t on human plasma after in vivo administration. h urb etrmore, ll 'asd gen lel tpllerzmen a Yt 2O Platelet aggregation studies have been performed on t e S ances re Q y p a e e :aggrega es f 1m rabbit or human platelet-rich plasma at a constant temate a process leading to the formation of new intravasperature of 37C, using the turbidometric method of cult" Plateet ,aggregate? w in mm, can Cause Born described in Nature (London), 194,927 I962). organ dysfunction, vessel in ury and thus further plate- Aggregation curves have been read fon'owing the let aggreganof" method described by OBrien et al.. Thromb. Diath.

A key step in stimulation of platelets is the interac- Haemorrhag 16 75] (1966). tion of the subendothelial tissue (collagen, basement In Order to Show the absence of ulcemgenic effects membrane, elastin) of the arterial wall with the plateof the compound 2 bis(lg hydroxyethyl)amin0 4,5 to comparison with the most widely known compounds sclerotic plaque. There is, therefore, strong evidence belonging to the Category of ami inflammmory drugs 3 3 1 i si; 5 623 8 f c gfizggn igfrgg Lhe inlductgon of gastric lgsiilriislin eclrperimental 21I'IlIll1lfllS 0 eta Them rant? 1 O u 0 as aso een eva uate ae istar rats weig ing brils, elastin, etc. will induce adherence of platelets ]70 190 were given the rested drugs orally for 17 lg t0 t l l i fihi j flggflttil Si l-fin an ef consecutjjve days after a daily fastlilng period of 8 hours. Tom 6 a 651 11 0 0V g When In omet acin was tested,t e drug was orally adfeCtiVe method Ofhlhibitlng Platelet aggregation Clearly ministered for 1 day only to the animals which had pp Platelet aggregation is inhibited 3g number been kept fasting for 18 hours. At the end of the experi- Of su sta s- However, the use of these 5U stances as ments, all of the surviving animals were sacrificed, their antlagglegatlng and Potential anti'thronlbotilf agents is 40 stomachs removed and microscopically examined for often limited by theirunacceptable in vivo side effects the presence f any l i on h gastric ll r undesirable biological actwltles- As shown by the following Table I, acetylsalicylic Among the Substances Wh1ch have been Shown to be acid, Indomethacin and, to a lesser extent, phenylbutausefuldfor the i l therapdeutlc a lg are i t t l zone exhibit the ability to inhibit collagen and adrena- Stefoi ammatory 2 h Oftunate y 659 line induced platelet aggregation. However, no signifidrugs are neither Capable of inhibiting Platelet g cant effects can be found on ADP-induced platelet agtion in all of its forms, nor are they free from severe unri s di saheylate h no k bl idesirable side effects. In fact, although the non-steroid, aggregating activity, anti-inflammatory drugs are effective in inhibiting the 0 h other h d Di l Shows a strong irelease of platelet constituents induced by collagen, 5O aggregating i i on b th d li d ll gamma-globulin Coated Surfaces, antigen-antibody induced platelet aggregation and, in addition, an appreomp x thrombine and adrenaline. y do not ciable effect on ADP induced platelet aggregation. hibit the primary platelet aggregation, that that Acetyl salicylic acid, Indomethacin, phenylbutazone duced by ADP. and sodium salicylate induce gastric haemorrhagic le- Moreover, it is Well known that most of the antisions when administered at the same dose levels as are inflammatory drugs induce gastric haemorrhagic leeffective in the usual anti-inflammatory tests. On the sions and ulcers. Therefore, the need exists of a new contrary, Ditazole does not display any significant ulcdrug which is capable of inhibiting ADP, collagen, er-inducing activity even at higher doses.

TABLE I INHIBITION OF PLATELET AGGREGATION INDUCED BY COMPOUND INDUCTION OF A D P Collagen Adrenaline GASTRIC LESIONS Acetyl Salicylate Acid Ditazole lndomethacin Phenylbutazonc Sodium Salicylate TABLE I -,C,ontinued INHIBITION OF PLATELET AGGREGATION INDUCED BY COMPOUND INDUCTION OF A D P Collagen Adrenaline GASTRIC 1 LESIONS of ulcerated rats active at: active at: active at: =2Xl0 M or above l-'l0"M or above l 0M or above I up to 33.3% H- =l0 M or above H-=5 l0 M or above lll0M or above 4+ up to 66.6% inactive -H+=IO"=M or above H+ =5 l0* M or above H+ up to 99.9% lll+=5 l0 M or above inactive Adenosine ll Further in vitro platelet aggregation studies after in vivo administration. to humanswere carried out as follows: Apparently healthy volunteers of 24 to 55 years of age, who had not received any drugs for at least 7 days prior to the study, were investigated in the fasting state. v v

Twelve subjects received 800 mg. of Ditazole daily for 2 days. Measurements'were made both before and 2 days after the start of drug administration.

Anothengroup of four subjects received only placebo. Measurements were made as indicated above for the first group. Slope and maximum transmission were recorded both before and after the drug administration, and expressed as change of the after measurement with respect to the before measurement of the same parameter. I

In the case of collagen-induced platelet aggregation, the delay time (reaction time") in seconds from the addition of the aggregating agent to the inflection of the curve was also measured and expressed as change as above.

Negative figures in slope and maximum transmission changes indicate that anti-aggregating activity is present; positive figures in reaction time changes indicate that the compound is effective in prolonging the reaction time in the collagen-induced platelet aggregation test.

The results are summarized in Tables 2 and 3.

TABLE 2 COLLAGEN (80 -g/ml) INDUCED PLATELET AGGREGATION IN HUMAN PLASMA MAX. REAC- TION NO. OF SLOPE, TRANSMISSION, TIME. DRUG SUBJECTS 7t 72 CHANGE CHANGE CHANGE DITAZOLE 6 -24.56 +1.95 +l 19.00 PLACEBO 4 +I5.30 +7.98 +l5.87

Acute toxicity has been determined on Ditazole, phenylbutazone and indomethacin in mice and rats by oral and i.p. administration. The results'are givenin Table 4.

TABLE 4 The above data clearly indicate that Ditazole is free from side effects, has a low toxicity and is a potent inhibitor of platelet aggregation. This evidence also suggests that the above compound is a potentially useful anti-thrombotic agent.

The compound useful in the practice of this invention can be formulated into suitable pharmaceutical dosage forms, e.g., coated or uncoated tablets, capsules, syrups, suspensions, suppositories, etc., by combining it with pharmaceutically acceptable carriers or diluents, in accordance with methods well known in the art. Suitable examples of dosage formulations useful in the practice of this invention are as follows:

EXAMPLE 1 Capsules containing 200 mg. of the active substance and having the following composition were prepared:

Ditazole 200 mg. glycocoll mg. starch 50 mg. talc 10 mg. magnesium stearate 10 mg.

:EXAMPLE 2 Suppositories were prepared, according to methods well known in the art, having the following composition: I i

Ditazolc 400 mgv Polyoxyethylcncglycol 6000 345 mg. Polyoxycthylencglycol 1540 805 mg.

1550 mgv A therapeutically effective daily dose in man has been found to be approximately 800 mg. Preferably, the drug is orally administered.

What I claim is:

l. A method of inhibiting platelet aggregation comprising the administration to man or animal in need of such treatment of a therapeutically effective amount of 2-bis(,B-hydroxyethyl)aminc-4,S-diphenyloxazole. 

1. A METHOD OF INHIBITING PLATELET AGGREGATION COMPRISING THE ADMINISTRATION TO MAN OR ANIMAL IN NEED OF SUCH TREATMENT OF A THERAPEUTICALLY EFFECTIVE AMOUNT OF 2-BIS(B-HYDROXYETHYL)AMINO-4,5-DIPHENYLOXAZOLE.
 2. The therapeutic method of claim 1 in which 2-bis( Beta -hydroxyethyl)amino-4,5-diphenyloxazole is administered in combination with a pharmaceutically acceptable carrier or diluent.
 3. The therapeutic method of claim 2 in which the 2-bis( Beta -hydroxyethyl)amino-4,5-diphenyloxazole is orally administered.
 4. The therapeutic method of claim 3 in which the 2-bis( Beta -hydroxyethyl)amino-4,5-diphenyloxazole is orally administered in the form of a capsule thereof.
 5. The therapeutic method of claim 1 wherein said therapeutically effective amount is approximately 800 mg per day. 